High TWIST1 mRNA expression is associated with poor prognosis in lymph node-negative and estrogen receptor-positive human breast cancer and is co-expressed with stromal as well as ECM related genes

Abstract

Introduction: The TWIST homolog 1 (TWIST1) is a transcription factor that induces epithelial to mesenchymal transition (EMT), a key process in metastasis. The purpose of this study was to investigate whether TWIST1 expression predicts disease progression in a large breast cancer cohort with long-term clinical follow-up, and to reveal the biology related to TWIST1 mediated disease progression.

Methods: TWIST1 mRNA expression level was analyzed by quantitative real-time reverse polymerase chain reaction (RT-PCR) in 1,427 primary breast cancers. In uni- and multivariate analysis using Cox regression, TWIST1 mRNA expression level was associated with metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS). Separate analyses in lymph node-negative patients (LNN, n = 778) who did not receive adjuvant systemic therapy, before and after stratification into estrogen receptor (ER)-positive (n = 552) and ER-negative (n = 226) disease, were also performed. The association of TWIST1 mRNA with survival endpoints was assessed using Kaplan-Meier analysis. Using gene expression arrays, genes showing a significant Spearman rank correlation with TWIST1 were used to identify overrepresented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-annotated biological pathways.

Results: Increased mRNA expression level of TWIST1 analyzed as a continuous variable in both uni- and multivariate analysis was associated with shorter MFS in all patients (hazard ratio (HR): 1.17, 95% confidence interval, (95% CI):1.09 to 1.26; and HR: 1.17, 95% CI: 1.08 to 1.26; respectively), in LNN patients (HR: 1.22, 95% CI: 1.09 to 1.36; and HR: 1.21, 95% CI: 1.07 to 1.36; respectively) and in the ER-positive subgroup of LNN patients (HR: 1.34, 95% CI: 1.17 to 1.53; and HR: 1.32, 95% CI: 1.14 to 1.53; respectively). Similarly, high TWIST1 expression was associated with shorter DFS and OS in all patients and in the LNN/ER-positive subgroup. In contrast, no association of TWIST1 mRNA expression with MFS, DFS or OS was observed in ER-negative patients. Genes highly correlated with TWIST1 were significantly enriched for cell adhesion and ECM-related signaling pathways. Furthermore, TWIST1 mRNA was highly expressed in tumor stroma and positively related to tumor stromal content (P <0.001).

Conclusions: TWIST1 mRNA expression is an independent prognostic factor for poor prognosis in LNN/ER-positive breast cancer. The biological associations suggest an involvement of the tumor microenvironment in TWIST1's adverse role in breast cancer.

Figure 1

Study design, patient inclusion criteria and patient sub-cohorts analyzed. Associations of TWIST1 mRNA expression levels with tumor aggressiveness were evaluated in all patients and in LNN, LNN/ER-positive and LNN/ER-negative patients. The LNN patients in this study did not receive adjuvant systemic therapy.

Figure 2

Kaplan-Meier survival curves presenting the association of TWIST1 mRNA expression with metastasis-free survival in: A: all 1,427 patients; B: LNN patients only, C: LNN and ER-positive patients and D: LNN and ER-negative patients. The LNN patients in this study did not receive adjuvant systemic therapy. The patients are divided into four quartiles (Q1 (low) to Q4 (high)) based on TWIST1 mRNA expression levels after normalizing these to the expression of our set of three reference genes (B2M, HMBS and HPRT1). Patients at risk at various time points are indicated.

Figure 3

Association of TWIST1 mRNA expression with stromal content of the tumor tissue. A: TWIST1 mRNA expression levels in laser capture microdissected breast tumor tissues. The stromal and epithelial tumor cell areas were separately analyzed after microdissecting 10 breast tumor tissues (n = 9 with high ESR1expression, n = 1, (T6), with low ESR1 expression). After LCM, RNA isolation and cDNA synthesis were done as described in the Patients and Methods sections. TWIST1 mRNA levels were measured by qRT-PCR in RNA isolated from both the stromal (S) and the epithelial tumor cell (T) compartments of these tissues, and levels were normalized to our set of reference genes by the delta (Δ) Ct method. To ensure our set of reference genes did not cause a bias in these analyses, and our LCM material was indeed enriched for stromal and epithelial cells, we also measured VIM and EpCAM in these fractions. A two-sided paired t-test was performed to evaluate the differences. B: TWIST1 mRNA expression in SR and SP ER-positive tumors and C: TWIST1 mRNA expression in SR- and SP ER-negative tumors. For figure sections B and C, the mRNA expression of TWIST1 was log-transformed and the expression relative to reference genes is presented on the y-axis of the box-and-whisker plots. The box-plot shows the five statistics (box bottom-line is the 25^th percentile, solid line in the middle of the box presents the median, upper line of the box is the 75^th percentile and the whiskers extend to 1.5 times the inter-quartile range, observations beyond these values are plotted, separately). Mann-Whitney test was performed to evaluate the difference between the SR and the SP groups in both ER-positive and ER-negative patients (B, C).

Figure 4

Representative tissues with IHC staining of TWIST1 protein. A: Strong nuclear staining of TWIST1 protein in an ER-positive tumor tissue with high TWIST1 mRNA expression. B: Weak nuclear staining of TWIST1 protein in an ER-negative tumor tissue with low TWIST1 mRNA expression. C: Correlation of TWIST1 mRNA expression and protein expression in breast tumor tissues (n = 20; n = 10 high TWIST1 mRNA expression, n = 10 low TWIST1 mRNA expression). Red and green boxes represent high and low TWIST1 mRNA expressing tumor tissues, respectively, whereas empty boxes and filled boxes represent ER-negative and ER-positive tumor tissues, respectively. Spearman rank correlation test was used to evaluate the correlation between TWIST1 mRNA and protein expression. D: Association of stromal staining of TWIST1protein with ER-positive tumor tissues. (▽ = negative nuclei of inflammatory cells; ▼ = strong positive nuclei of inflammatory cells; ↕ = strong positive nuclei of fibroblast; ⇨ = strong positive nuclei of tumor cells; ↓ = strong positive nuclei of endothelial cells).